DESCRIPTION: The overall objective of this program remains to understand specific protein-DNA recognition and binding dynamics in solution. NMR spectroscopy and genetic engineering methods are being used to characterize structural, functional, and dynamic features of this interaction. The biological system under investigation is the methionine aporepressor protein dimer, metJ, its corepressor S-adenosylmethionine, and synthetic fragments of the met operator DNA. Based on recent crystallographic data, this repressor protein represents a novel DNA binding motif involving insertion of two antiparallel strands, one from each protein monomer, into the major groove of the duplex DNA and provides a complete and tractable system for detailed NMR investigation. The overall effort is designed to generate a better understanding of how these interactions control the expression of genes in the methionine regulon. To achieve these objectives, heteronuclear and homonuclear, multidimensional NMR methods are being developed and used along with spectral editing techniques to study solution dynamics and structures of the protein and its complexes. The mechanism by which the corepressor enhances DNA binding and repression is of particular interest, since it appears to influence binding from long distances in the crystal structure. Methods to examine solvent exposure in the protein and protein-DNA complex via fast proton exchange are being developed to provide insight into the dynamic behavior of the system in solution. Protein backbone dynamics are also being explored in the free and bound protein using heteronuclear magnetization relaxation methods. MetJ labeled with the stable isotopes C-13, N-15, and H-2 is being used in these studies. The DNA binding characteristics of mutant proteins selected, based on results of gel shift and Dnase protection screening, will also be studied by NMR methods.